The channels that comprise gap junctions, provide a conduit for diffusion of small molecules between neighboring cells. Two hemichannels, or connexons, comprise each gap junction channel; each connexon is composed of six subunits called connexins (Cx). The mammalian genome contains at least fifteen different Connexins, four of which are expressed in the cardiovascular system (Cxs 43, 40, 45 and 37). Gap junctions make coordinated contractile activity of the heart and blood vessels possible by providing a pathway for electrical communication. They also play important roles in growth and development. Cxs 40 and 43 form homomeric/homotypic channels (all connexins in the channel are identical) with very different functional properties. However, Cxs 40 and 43 are rarely expressed in isolation. They are co- expressed at ratios that vary as a function of development (and aging), injury, and disease in ventricular and atrial tissue as well as in vascular endothelium and smooth muscle. Cxs 40 and 43 form functional "mixed" channels (heteromeric/heterotypic) with unknown properties. The long-term goal of the proposed studies is to determine how Cx40-43 comprised junctions contribute to cardiovascular development, injury and disease. Our specific aims include the following goals. 1) Determine whether long-term changes in Cx40: Cx43 expression ratio cause predictable changes in junctional permeability and cellular growth properties. 2) Assess whether the magnitude of the acute decrease in junctional conductance induced by PDGF depends on the Cx40: Cx43 ratio and whether junctional permeability decreases concomitantly. 3) Examine the role of Cx43 phosphorylation in these acute effects of PDGF. And 4) determine the time course and underlying mechanisms for recovery from the acute effects of PDGF. Smooth muscle cells induced to express varied Cx40: Cx43 ratios naturally and through genetic manipulation and Rin cells transfected with these genes will serve as model systems for these studies. Gap junction permeability and channel function will be assessed using dual whole cell and perforated patch voltage clamp techniques. Cell proliferation is evaluated using flow cytometry to assess cell cycle behavior of the cells. Connexin expression and distribution are evaluated using 3-D imaging of immumo-stained cells and quantitative Westerns. This powerful combination of approaches offers a unique opportunity to discover how gap junctions contribute to development, injury and disease in the heart and vasculature.